A home and rescue gene drive efficiently spreads and persists in populations

  1. Nikolay P. Kandul
  2. Junru Liu
  3. Jared B. Bennett
  4. John M. Marshall
  5. Omar S. Akbari

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Abstract

Homing based gene drives, engineered using CRISPR/Cas9, have been proposed to spread desirable genes into target populations. However, spread of such drives can be hindered by the accumulation of resistance alleles. To overcome this significant obstacle, we engineer an inherently confinable population modification Home -and- R escue (HomeR) drive in Drosophila melanogaster that, by creative design, limits the accumulation of such alleles. We demonstrate that HomeR can achieve nearly ∼100% transmission enabling it to spread and persist at genotypic fixation in several multi-generational population cage experiments, underscoring its long term stability and drive potential. Finally, we conduct mathematical modeling determining HomeR can outperform contemporary gene drive architectures for population modification over wide ranges of fitness and transmission rates. Given its straightforward design, HomeR could be universally adapted to a wide range of species.

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  1. eLife

    Reviewer #4:

    General assessment of the work

    Gene drives can be used for sustainable control of disease vectors, and there is a need for a different gene drive strategies that can be tailored to the particular species, timescale, and desired spatial spread. Kandul and colleagues present a welcome new addition to the growing number of strategies for gene drive, called HomeR, that combines elements of killer-rescue and homing-based drive to exert spatiotemporal control over its spread, whilst counteracting the rise of resistant mutations. Whilst it is extremely promising, some major claims of this manuscript are inaccurate or unsupported by the evidence. The authors could easily address the most important concerns by expanding their sequencing analysis to better detect and quantify resistant mutations, paying careful attention not to overstress the potential of this drive to mitigate resistance, and by comparing the relative strengths of different drive strategies instead of focussing only on features that are most flattering to the HomeR strategy.

    Numbered summary of any substantive concerns

    1. The drive release strategy of Fig 4A + 4C is primed to underestimate and potentially mask resistance. In Fig 4A, where the authors search for signs of resistance, the population was seeded with males that were all homozygous for the drive, meaning that 100% of their G0 progeny will inherit it. As the rate of homing is close to 99%, only a small fraction of their G1 could have inherited a non-drive (potentially resistant allele) allele. In a realistic release scenario, resistant alleles will have ample opportunity to be generated and subsequently selected. Though still far from adequate, resistance testing would have been better performed on samples collected from the lower frequency releases in panel C. This experiment should not be used to draw strong conclusions about resistance to pHomeR, but should be used to make broader observations regarding the spread and stability of the construct.

    2. The strategy for sampling resistance will obscure almost all resistance in the population, and would fail to detect even a strong selection for it. Flies were only selected for resistance genotyping if they lacked GFP, meaning they carry two non-HomeR alleles (i.e. homozygous for the R1 allele or transheterozygous with another R1/R2/WT). One would expect most resistant alleles to be heterozygous in a population that was seeded with almost complete drive homozygosity. The authors could, and should, have done more to identify and quantify these. Amplicon sequencing was used to sample the full diversity of alleles in a larger pool of individuals (including GFP+ flies) collected at G10, why was this approach not used throughout? By adopting the approach earlier they would have been able to track the changing frequencies of R1 and R2 alleles over time.

    3. The impression given in the figure and main text is that R1 alleles were rare (or entirely absent), when they were not. In spite of the incredible advantage given to the drive, and a bias in sampling method that would mask the presence of resistant alleles, resistance was observed in every generation tested (G2, G3 and G10). The authors claim that because GFP-individuals were not observed in later generations, the resistant alleles had not come under positive selection. This logic is flawed, and indeed their own amplicon sequencing analysis performed on G10 flies revealed several resistant alleles, including an R1 present in 80% of non-drive alleles. The two most frequent mutant alleles detected were in frame, and I do not agree that these are likely to be deleterious recessive (as the authors speculated). These could be functionally resistant mutations. I believe there were many more R1 alleles in heterozygosity with the HomeR allele, these alleles could have been spreading, but were excluded from the genotyping analysis. Could these putative R1 individuals not have been specifically tested to see if they do, or do not confer resistance?

    4. The modelling takes a very limited approach to comparing different drive strategies, and by comparing proof-of-principle designs, important differences are obscured. For example, simple modifications that would mitigate resistance are likely to be included in many designs - such as multiplexing gRNAs. The nuances of each design are lost in a discussion focused on the rate of spread, which is largely irrelevant now because all of the drives are predicted to spread well.

    5. The authors did not discuss the relevance of having performed releases in a population that was already homozygous for Cas9. Do the release experiments and model really suggest the drive could spread if released into an otherwise WT population? I'm not sure the data presented in this manuscript can support that claim.

  2. eLife

    Reviewer #3:

    The authors are to be commended for the effort put into careful experimental design and clear presentation of methods and results.

    My main concern with the manuscript is that the claim about their specific polymerase gene being "ultraconserved" is not backed up with their own data or by citations from the literature. If the gene sequence was ultra-conserved, I wouldn't have expected the authors to be able to do so much recoding of the gene without fitness consequences. Furthermore, it is clear that homozyogous-viable NHEJ mutations did develop in the experiment. Without explanation, this seems to be a fatal flaw in the design.

    This manuscript describes a modification of the general homing gene drive concept by use of a split drive system that increases the frequency of a recoded polymerase gene that replaces a cleavage susceptible, naturally occurring, haplosufficient, conserved polymerase gene. This approach is taken in order to limit the evolution of cleavage resistance in the naturally occurring gene.

    I am not convinced that the research presented achieves the intended goals. I did a quick look for literature on the "ultraconserved" polymerase pol-y35 gene and could find none. I am not sure if the conservation is at the DNA sequence level or at the amino acid level. If at the amino acid level, then it makes sense that resistance alleles can form at the DNA level that don't impact the protein at all. Figure 2a shows the 22 and 27 recoded nucleotides for the two guide RNA sites. The authors say that these changes to the sequences didn't seem to impede fitness. Did the authors try many other recodings and finally decide on these because all others caused loss of fitness, or is it just that this gene is robust to substitutions even though the protein is conserved.

    Figure 4C shows that the frequency of flies with at least one copy of the pol-y35home R1 increased from about 25% to about 50% between the parental and F0 generation when there was no Cas9 present. As long as the transgenic males were competitive with the wild flies this makes sense because the released flies were homozygous for that allele and the offspring should all have inherited one copy of the gene. What doesn't make sense is that when the work was done with all flies harboring the Cas9, the pol-y35home R1 increased less than in the former case, from the parental to generation F0, the frequency of flies with the pol-y35home R1. In some replicates the frequency of such flies didn't increase at all. It should be noted that the parents were always homozygous. This certainly indicates a fitness cost to the flies with a combination of Cas9 and the homing construct.

    In this same figure, results from the model are plotted. It seems like the model assumes no fitness cost because it shows an exact increase from 25% to 50% flies carrying at least one copy of the pol-y35home R1 theoretical construct. In later generations the experimental results outperform the model. Presumably, this model is used to construct figure 6. This mismatch needs to be addressed in the manuscript.

    The fact that in all three replicates of the experiment without Cas9, the F0 is above 50% indicates that something else may be going on that is unrelated to gene drive. It could be due to heterosis between the two slightly different strains of flies. When wildtype males mate with wildtype females, the offspring are more inbred than when a transgenic male mates with a wildtype female. Just a hypothesis.

  3. eLife

    Reviewer #2:

    Kandul et al. present an interesting study that could lead to important improvements on the use of homing-based gene drives. However, there are a number of things that should be addressed to improve the manuscript for better comprehension by readers.

    Overall the manuscript presents a load of data. But the presentation of these data could be made in a better digestible way. The authors should go over their manuscript with a reader in mind, that is interested but not necessarily knows all the relevant literature in the very detail.

    Abstract (line 18): Please remove "inherently confinable" from the abstract. The drive is indeed designed in a split drive design, however, all the experiments were done in a homozygous Cas9 background. Therefore, there are no experimental data for a split drive provided in this manuscript. The split situation seems to be here more for a practical reason to be allowed to do the experiments in a less stringent laboratory environment. Thus there are no experimental data that would support the confineable nature of this drive. Actually there are not even modelling data to this. Thus, such a statement should not be put in the abstract. This manuscript is not a demonstration of a confineable drive.

    Results (line 124): How was Pol-gamma35 identified? It would be interesting to the reader to get to know about the exact reasoning, why this gene was chosen. Or were there several ones chosen before and this turned out to work the best or was the easiest to design. This could be very interesting considerations important to the field.

    Results (lines 147-148 Fig. 1B; lines 155-156 Fig. 1C) and Methods (lines 698 and 706) and Figure 1 (both Figure and legend): The addressing of the Figure panels and the writing to it don't fit! Has there been a rearrangement of the Figure that was not worked through the text? When referring to "B" in the text, it is still about Act5C-Cas9 and the nos-Cas9 data are in the text referred to Fig1C! But Fig1C is BLM! In current panel Fig1B, what does "all" mean below the X-axis? This is not comprehensible. Panel C is not really described in the Figure legend!

    Results (line 253), Discussion (lines 526-527), and supplementary Figure 1 (line 1101). "converting recessive non-functional resistant alleles into dominant deleterious /lethal mutations" is completely misleading! There is no "conversion" and how should that be done molecularly. There is a continuous removal of such alleles from the population because of lethal transheterozygous conditions caused in the drive. However, there is no active conversion of such alleles into dominant lethal ones. This needs to be clearly rewritten to avoid the misleading idea. Supplementary Figure 1 also seems to have a slight conceptual problem. What are "cells" (rectangles) with a red frame and a green core? Green means at least one wt allele (this must include the recoded rescue allele!). Red means biallelic knock-out: thus a red cell cannot have a wt allele. Thus what is a red-framed green core cell? To explain the removal of R2 alleles, a depiction of yellow framed red core cells in the germ line would be helpful, since this would explain how R2 alleles are selected against and might be continuously removed from the population!

    Results (from line 424 to end of results): Before going into the modelling, the reader should be clearly informed about all the different approaches that are now to be compared. This is currently not done well, if at all! Thus moving current Fig 6 before current Fig. 5 might clearly help! Also a better explanation of the panels in Figure 6 is necessary as well as a correction of Fig6 Panel E! A comparison of a great number of the currently approached toxin-antidote (gene destruction - rescue, but not killer-rescue!) systems is greatly appreciated. However, the authors cannot expect the general reader to know about the small detailed differences between the systems that are compared here. Thus the authors need to provide some explanations and categorization of the different approaches here and also cite all the respective literature.

    -First subdivision: Non-homing (interference-based drives) VERSUS Homing (thus overreplication-based drives). This will also help them to better understand why the interference-based drives (TARE and ClvR) are more sensitive to fitness parameters than overreplication drives.

    -Second subdivision: same-site VERSUS distant site. This is important to understand the difference between the here modelled TARE and the CLvR. Actually ClvR is a TARE, but you use TARE here more specifically as the results in the respective paper are demonstrating only a same-site TARE! But this needs to be clearly stated here!

    -Third subdivision: viable VERSUS haplosufficient VERSUS haploinsufficient. This also needs to be clearly depicted in labelling panels C to F of Figure 6, which are currently hard to grasp what the essential differences are, before looking at the panels in detail: C: HGD of viable gene (HGD) D: HGD of viable gene with rescue (HGD+R) E: HGD of haploinsufficient gene with rescue (HGD-hi+R). THIS PANEL NEEDS MAJOR CORRECTION!! F: HGD of haplosufficiant (essential) gene with rescue (HomeR)

    -Forth subdivision: split VERSUS non-split. Here for the split HGD situation, the respective papers of which the current authors are co-authors should be cited: Kandul et al. 2020 (actually published end of 2019 and still cited as biorxiv Archives article 2019a!?) and Li et al. 2020, Elife). In addition, it is also important to state clearly that "split or two locus" is completely independent of the "distant site" concept! The reader needs to understand the differences of the systems that are compared here, without having the reader to go to the respective publications themselves and then try to find out what the differences really are. This is not so obvious and the current authors have a clear chance here to do that and help the reader in the mists of all this similar but still distinct approaches.

    Figure 6 Panel E: This depiction is not consistent within itself, not consistent with the legend, and not consistent with the cited literature!

    -Why should the rescuing drive construct over the wt allele be lethal as indicated in the right two boxes?

    -The cited paper Champer et al. 2020b, which is by now also published in PNAS! clearly states that there is maternal carry over, which actually makes it so hard to use and is probably only working via male propagation. In the Figure legend, it is said that "maternal carryover and somatic expression ... are empirically unavoidable", which is in contrast with the depiction! The legend then also states that this is "unachievable". This should be better replaced by "hard to achieve", since the approach is published and seems to drive, even though probably just via the males! Thus the depiction of panel E needs to be thoroughly revised.

    Discussion (lines 499-500): The haplolethal HGD works (admittingly poorly) despite the maternal carryover (Champer et al 2020b). Therefore, your statement needs to be refined or deleted: "requires germline-specific promoter that lacks maternal carryover" is not consistent with the published paper! The drive could go via the males because then you do not have maternal carry over! And homing based drives can go via males and do not necessarily have to be promoted through females, see also KaramiNejadRanjbar et al. 2018.

    Discussion (lines 540 to 543). This sentence is based on an old but clearly overruled idea! NHEJ repair is not restricted to a time before the fusion of the paternal and maternal genetic material. It has been clearly demonstrated that R1 and R2 alleles are also generated in the early embryo after the zygote state (Champer et al 2017, KaramiNejadRanjbar et al. 2018). Actually, all of the authors' Figure 1C and Supplementary Figure 1 are about NHEJ mutation in the early embryo causing "BLM". Thus this sentence is inconsistent with current beliefs and also with the authors' own writing!

    Figure 4: Panel C graph: Why is, in the controls, the transgene consistently and significantly higher inherited to the next generation (0). It is about 75% progeny sired by the transgenic fathers compared to the wild type fathers? Was there an age advantage of the transgenic ones or whatever other fitness factor? This is surprising and no explanation is given at all! In contrast, in the Cas9 background in generation 0, less than 50% carry the drive allele, which is probably due to induced lethality. But this should also be commented upon. In the legend it is stated that 7 of 9 flies carried an R1 allele heterozygous to an R2 allele. What about the other two?

  4. eLife

    Reviewer #1:

    This paper shows that Cas 9 mediated homology directed repair can be used to insert a synthetic rescue gene into an essential gene, here mitochondrial Pol-gamma35 was chosen. The insertion is marked by an eyeless-GFP reporter and also contains the gRNA (gene drive) but not the Cas9 (considered as a safe split gene drive). 'Homing' of the eye-GFP is assayed to detect insertion at the homologous locus when Cas9 is present by HDR. The authors show that this works well in the female germline with various tested Cas9 lines (vas, nos, Act5C and ubiq-Cas9). In all cases close to 100% transmission to the homologous locus on the homologous chromosome is achieved when an effective guide RNA is used. Hence, eye GFP transmits ('homes') in a 'super-Mendelian' ratio at the chosen target. A male specific transmission works less well (exuL-Cas9). The reason why it works well appears to be that the chosen target is an essential gene (Pol-gamma35) in which small changes caused by NHEJ that result in homing 'resistant' alleles will be loss of function alleles and hence will not spread in the population. Unfortunately, the authors did not test how the drive could spread in a wild type population (no Cas9 expression). I am also missing a test relevant for pest studies that would achieve the spread of a potentially deleterious or beneficial insertion that could kill a population or make it resistant to a disease.

    1. This paper is very hard to read. Sentences are excessively long and complicated. References to the Figures appear not always correct.

    2. Figure 1. Genotypes in Figure 1A are unreadable in the print version because of the small font. Are the 2 crossing schemes required that only differ in gRNA1 or gRNA2? The surviving progeny should be quantified as in Fig 1B. Figure 1B shows nos-Cas9 and not act-Cas9 results (several typos in line 148-155). Figure 1C: the incidence of heterozygous, homozygous and 'resistant' cells is schematic and not supported by data, hence questionable if Figure 1C should be shown in results.

    3. Figure 2. Genotypes not readable in print. Is it necessary to show schemes of the procedure how transgenic flies were generated and how the Pol-gamma 35 HomeR were made with all chromosomes detailed (Fig 1D)? This could move to the methods as it is standard and we learn not much new.

    4. More typos: line 286: Fig2B is the wrong reference; line 295 should read Actin 5C. Figure 4B GGG codes for Gly (not Gla). lines 576 to 592 should refer to Figure 6?

    5. Figure 5 - as figures 1+ 2, only readable on the computer.

    6. It would be interesting to see how the gene drive would spread if Home R and Cas9 would be introduced in a competitive way into wild type populations. This is similar to Fig 4C, but the only the Home R males or females would carry the Cas9. This would be a more realistic test how the gene drive could spread in a wild population that obviously does not express Cas9.

  5. Version published to 10.1101/2020.08.21.261610v4 on bioRxiv
  6. Version published to 10.1101/2020.08.21.261610v3 on bioRxiv
  7. Version published to 10.1101/2020.08.21.261610v1 on bioRxiv
  8. Version published to 10.1101/2020.08.21.261610v2 on bioRxiv